Liquid chromatography–tandem mass spectrometry method for quantification of thymidine kinase activity in human serum by monitoring the conversion of 3′-deoxy-3′-fluorothymidine to 3′-deoxy-3′-fluorothymidine monophosphate

Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3′-deoxy-3′-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3′-deoxy-3′-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography–MS/MS (LC–MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5–500 ng/mL for FLT and 2.5–2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.

► A LC–MS/MS for measuring thymidine kinase activity in human serum is described.
► Phosphorylation of 3′-deoxy-3′-fluorothymidine indicates activity.
► The monophosphorylated product is measured quantitatively.
► The method uses protein precipitation and on-line column switching.
► The method was FDA validated and applied to hepatocellular carcinoma patients.